The need for a study of the stability of physostigmine in fresh human plasma and whole blood became apparent upon reviewing the literature. A High Performance Liquid Chromatography (HPLC) method utilizing fluorescence detection was developed for the quantitation of physostigmine in plasma, whole blood and CSF. The degradation of physostigmine under various controlled conditions was documented at time 0, 10, 20, 30, 60 minutes. The mean half-life of physostigmine in fresh human plasma at 25 degrees C and 37 degrees C was 28.9 and 26.6 minutes respectively. The half-life of physostigmine in stored (blood bank) plasma at 25 degrees C and 37 degrees C was 69 and 24 minutes, respectively. The best method for the collection of clinical samples containing physostigmine was to place the sample into a cold (0 degrees C) tube containing neostigmine bromide (5 mg/ml) and place the tube immediately on ice. This method was used to document the disposition of physostigmine.